Figure 12

Lentivirus designed with a 1.3 αCaMKII promoter and an intron conferred in vitro and in vivo expression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).